Simultaneous assay of Src SH3 and SH2 domain binding using different wavelength fluorescence polarization probes

被引:15
作者
Lynch, BA [1 ]
Minor, C [1 ]
Loiacono, KA [1 ]
van Schravendijk, MR [1 ]
Ram, MK [1 ]
Sundaramoorthi, R [1 ]
Adams, SE [1 ]
Phillips, T [1 ]
Holt, D [1 ]
Rickles, RJ [1 ]
MacNeil, IA [1 ]
机构
[1] ARIAD Pharmaceut Inc, Cambridge, MA 02139 USA
关键词
fluorescence polarization; SH3; SH2; Src; binding assay; p130(CAS);
D O I
10.1006/abio.1999.4305
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
pp60(c-src) is a prototypical nonreceptor tyrosine kinase and may play a role in diseases as diverse as cancer and osteoporosis. In Src, the SH3 domain (Src homology 3) binds proteins at specific, proline-rich sequences, while the SH2 domain (Src homology 2) binds phosphotyrosine-containing sequences. Inhibition of Src SH3 and SH2 domain function is of potential therapeutic value because of their importance in signaling pathways involved in disease states, We have developed dual-wavelength fluorescent peptide probes for both the Src SH3 and the Src SH2 domains, which allow the simultaneous measurement of compounds binding to each domain in assays based on the technique of fluorescence polarization. We demonstrate the utility of these probes in a dual-binding assay (suitable for high-throughput screening) to study the interactions of various peptides with these domains, including a sequence from the rat protein pl30(CAS) which has been reported to bind simultaneously to both Src SH3 and SH2 domains. Utilizing this dual-binding assay, we confirm that sequences from pl30CAS can simultaneously bind Src via both its SH3 and its SH2 domains. We also use the dual-binding assay as an internal control to identify substances which inhibit SH3 and SH2 binding via nonspecific mechanisms. (C) 1999 Academic Press.
引用
收藏
页码:62 / 73
页数:12
相关论文
共 28 条
[1]  
Alexandropoulos K, 1996, SIN GENES DEV, V10, P1341
[2]   The formation of a covalent complex between a dipeptide ligand and the src SH2 domain [J].
Alligood, KJ ;
Charifson, PS ;
Crosby, R ;
Consler, TG ;
Feldman, PL ;
Gampe, RT ;
Gilmer, TM ;
Jordan, SR ;
Milstead, MW ;
Mohr, C ;
Peel, MR ;
Rocque, W ;
Rodriguez, M ;
Rusnak, DW ;
Shewchuk, LM ;
Sternbach, DD .
BIOORGANIC & MEDICINAL CHEMISTRY LETTERS, 1998, 8 (10) :1189-1194
[3]  
Bennett J., 1985, NEUROTRANSMITTER REC, V2nd, P61
[4]   Peptide ligands of pp60(c-src) SH2 domains: A thermodynamic and structural study [J].
Charifson, PS ;
Shewchuk, LM ;
Rocque, W ;
Hummel, CW ;
Jordan, SR ;
Mohr, C ;
Pacofsky, GJ ;
Peel, MR ;
Rodriguez, M ;
Sternbach, DD ;
Consler, TG .
BIOCHEMISTRY, 1997, 36 (21) :6283-6293
[5]   MODULAR BINDING DOMAINS IN SIGNAL-TRANSDUCTION PROTEINS [J].
COHEN, GB ;
REN, RB ;
BALTIMORE, D .
CELL, 1995, 80 (02) :237-248
[6]  
Dalgarno DC, 1997, BIOPOLYMERS, V43, P383
[7]  
FENG S, 1995, SCIENCE, V266, P1241
[8]   Specific interactions outside the proline-rich core of two classes of Src homology 3 ligands [J].
Feng, SB ;
Kasahara, C ;
Rickles, RJ ;
Schreiber, SL .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1995, 92 (26) :12408-12415
[9]   INACTIVATION OF THE PYRUVATE-DEHYDROGENASE COMPLEX OF ESCHERICHIA-COLI BY FLUOROPYRUVATE [J].
FLOURNOY, DS ;
FREY, PA .
BIOCHEMISTRY, 1989, 28 (25) :9594-9602
[10]   CALCULATION OF PROTEIN EXTINCTION COEFFICIENTS FROM AMINO-ACID SEQUENCE DATA [J].
GILL, SC ;
VONHIPPEL, PH .
ANALYTICAL BIOCHEMISTRY, 1989, 182 (02) :319-326